Microfabricated integrated dna analysis system

ABSTRACT

Methods and apparatus for genome analysis are provided. A microfabricated structure including a microfluidic distribution channel is configured to distribute microreactor elements having copies of a sequencing template into a plurality of microfabricated thermal cycling chambers. A microreactor element may include a microcarrier element carrying the multiple copies of the sequencing template. The microcarrier element may comprise a microsphere. An autovalve at an exit port of a thermal cycling chamber, an optical scanner, or a timing arrangement may be used to ensure that only one microsphere will flow into one thermal cycling chamber wherein thermal cycling extension fragments are produced. The extension products are captured, purified, and concentrated in an integrated oligonucleotide gel capture chamber. A microfabricated component separation apparatus is used to analyze the purified extension fragments. The microfabricated structure may be used in a process for performing sequencing and other genetic analysis of DNA or RNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S.application Ser. No. 12/844,544, filed Jul. 27, 2010 (Atty. Docket No.UCALP054C1), titled “MICROFABRICATED INTEGRATED DNA ANALYSIS SYSTEM”,which claims priority to U.S. application Ser. No. 11/139,018, filed May25, 2005 (Atty. Docket No. UCALP054), titled “MICROFABRICATED INTEGRATEDDNA ANALYSIS SYSTEM,” which is now issued U.S. Pat. No. 7,799,553, whichclaims the benefit under 35 U.S.C. 119(e) from Provisional U.S. PatentApplication No. 60/576,102, filed Jun. 1, 2004 (Atty. Docket No.UCALP054P), entitled “MICROBEAD INTEGRATED DNA ANALYSIS SYSTEM (MINDS),”all of which are incorporated herein by reference for all purposes.

STATEMENT REGARDING GOVERNMENTAL SUPPORT

The invention was made with government support under Grant NumbersAI056472, CA77664, and HG001399 awarded by the National Institute ofHealth. The government has certain rights in this invention.

BACKGROUND

1. Field of Invention

The present invention relates to microfabricated and microfluidicstructures. In one example, the present invention relates to amicrofabricated system and method for genome sequencing.

2. Description of Related Art

The genome of an organism is defined by the DNA (deoxyribonucleic acid)or, for some viruses, the RNA (ribonucleic acid) of the organism. Genomesequencing is figuring out the order of the nucleotides, or bases, of aDNA or RNA strand.

A current approach to genome-scale sequencing of DNA is shown in FIG. 1.This shotgun sequencing approach 100 uses bacterial transformation,selection and growth to manipulate individual genomic DNA fragments. Thefirst three steps of this approach, shearing, vector ligation, andtransformation (steps 102, 104, and 106), need only be performed once.As such, they do not present any particular problems. The last step, thecapillary electrophoresis (CE) (step 114), has been miniaturized throughmicrofabrication. This has reduced the cost and processing timeassociated with this step. The plate and grow (step 108), and the pick,grow and extract steps (step 110), however, are problematic. Thesesteps, which precede the Sanger extension step (step 112), perform cloneisolation and insert amplification. The amplification may be bacterial,PCR (polymerase chain reaction) or RCA (rolling circle amplification).These steps have remained refractory to miniaturization and integration.The current macroscopic paradigm has thus relied upon the use ofrobotics as the enabling technology for these key steps. However, thisis a problem because no less than 30 million colonies must be picked andgrown to produce a sequencing template for a genome. Furthermore, theminimum quantities of materials prepared by such robotic technologiesare orders of magnitude more than that required by modernmicrofabricated CE analysis systems.

Therefore, it is desirable to improve the processing time, volume scale,and level of integration of genome sequencing. It is also desirable toreduce the cost and space requirements of genome sequencing.

SUMMARY

In one aspect, the invention features a microfabricated structureincluding a distribution channel to distribute microreactor elementscarrying multiple copies of a clonal sequencing template into aplurality of thermal cycling chambers. Only one microreactor element ispassed into one thermal cycling chamber wherein thermal cyclingextension fragments are produced from a microreactor element.Purification chambers are connected to the thermal cycling chambers tocapture and concentrate the extension fragments. Component separationchannels are connected to the purification chambers to analyze theextension fragments.

Various implementations of the invention may include one or more of thefollowing features. The microreactor includes a microcarrier elementthat carries the multiple copies of the clonal sequencing template. Themicroreactor element is a bolus or microemulsion droplet. Themicroreactor element includes a microsphere carrying the multiple copiesof the clonal sequencing template. The sequencing template is a DNA orRNA sequencing template.

In yet another aspect, the invention is directed to a system forperforming sequencing. The system includes a means for shearing DNA orRNA into fragments and means for ligating the fragments to form amixture of desired circular and contaminating linear products. Thesystem further includes means for selectively removing the contaminatinglinear products and means for generating microreactor elements carryingmultiple clonal copies of a single sequencing template. The system alsoincludes means for selecting which microreactor elements have asequencing template and microfluidic distribution means for distributinga selected microreactor element with a sequencing template into athermal cycling chamber. Additionally, the system includes means forensuring that only one microreactor element will flow into one thermalcycling chamber and extension means, including the thermal cyclingchambers, for producing thermal cycling extension fragments from themicroreactor elements carrying multiple copies of the sequencingtemplate. Purification chamber means for capturing, purifying andconcentrating the extension fragments, and component separation meansfor analyzing the extension fragments are also part of the system.

Other implementations of the invention may include one or more of thefollowing features. The microreactor element includes a microcarrierelement that carries the multiple copies of the clonal sequencingtemplate. The microreactor element is a bolus or a microemulsiondroplet. The microreactor element includes a microsphere carrying themultiple copies of the sequencing template.

In another aspect, the invention features a microfabricated structureincluding a distribution channel to distribute microspheres carryingmultiple copies of a clonal sequencing template into a plurality ofthermal cycling chambers. An autovalve is located at an exit port of athermal cycling chamber to ensure that only one microsphere will flowinto one thermal cycling chamber wherein thermal cycling extensionfragments are produced from the microsphere. Purification chambers areconnected to the thermal cycling chambers to capture and concentrate theextension fragments. Component separation channels are connected to thepurification chambers to analyze the extension fragments.

Various implementations of the invention may include one or more of thefollowing features. The diameter of a microsphere is between about 1 and100 microns. The diameter of a microsphere is about 10 microns. Eachthermal cycling chamber is in fluid communication with a separationchannel. The sequencing template is a DNA or RNA sequencing template.

In still another aspect, the invention is directed to a microfabricatedstructure including a microfluidic distribution channel means fordistributing a microsphere carrying a sequencing template into a thermalcycling chamber. Autovalving means are used to ensure that only onemicrosphere will flow into one thermal cycling chamber. Extension means,including the thermal cycling chamber, produce thermal cycling extensionfragments from a microsphere carrying a sequencing template. Integratedpurification chamber means are used for capturing, purifying andconcentrating the extension fragments. Component separation means areused to analyze the extension fragments.

Other implementations of the invention may include one or more of thefollowing features. The extension means is a Sanger extension meansincluding a plurality of thermal cycling chambers. The autovalving meansincludes an autovalve at an exit port of the thermal cycling chambers.Purification chamber means includes purification chambers connected tothe thermal cycling chambers. The component separation means is acapillary array electrophoresis means including a plurality ofmicrochannels connected to the purification chambers. The sequencingtemplate is a DNA or RNA sequencing template.

In a further aspect, the invention is directed to a microfabricatedapparatus including a thermal cycling chamber. The thermal cyclingchamber is configured to receive a microsphere carrying a clonaltemplate. The chamber has an inlet port and an outlet port wherein theoutlet port includes a constriction that is configured to trap amicrosphere in the chamber and to substantially block further flow intothe thermal cycling chamber.

Various implementations of the invention may include one or more of thefollowing features. The shape of the constriction is substantiallycircular or semicircular. A first value is located in an inlet channelin fluid communication with the inlet port and a second valve is locatedin an outlet channel in fluid communication with the outlet port. Inoperation, the second valve is closed before the first valve to move amicrosphere out of the constriction and into a main body portion of thechamber before thermal cycling. A purification chamber is in fluidcommunication with the outlet port of the thermal cycling chamber and anoutlet port of the purification chamber is in fluid communication with acomponent separation apparatus.

In yet another aspect, the invention features a system for performingsequencing. The system includes means for shearing DNA or RNA intofragments and means for ligating the fragments to form a mixture ofdesired circular and contaminating linear products. The system furtherincludes means for selectively removing the contaminating linearproducts and means for generating microspheres carrying multiple clonalcopies of a single sequencing template. The system also includes meansfor selecting which microspheres have a sequencing template andmicrofluidic distribution channel means for distributing a selectedmicrosphere with a sequencing template into a thermal cycling chamber.Additionally, the system includes means for ensuring that statisticallyonly one microsphere will flow into one thermal cycling chamber. Thesystem also includes extension means, including the thermal cyclingchambers, for producing thermal cycling extension fragments from themicrospheres carrying multiple copies of the sequencing template.Purification chamber means for capturing, purifying and concentratingthe extension fragments, and component separation means for analyzingthe extension fragments are also part of the system.

Other implementations of the invention may include one or more of thefollowing features. The ensuring means is at least one of an autovalvein the thermal cycling chamber, an optical detector, and a timingmechanism. The optical detector is an optical scanner that detects lightscattered from a microsphere. The timing mechanism includes a pneumaticinput located adjacent to an inlet of a thermal cycling chamber.

In a further aspect, the invention features a system for performing DNAsequencing. The system includes means for shearing DNA into DNAfragments and means for ligating the DNA fragments to form a mixture ofdesired circular and contaminating linear products. The system alsoincludes means for exonuclease degredation for selectively removing thecontaminating linear products and emulsion PCR reaction means forgenerating microspheres carrying multiple clonal copies of a single DNAsequencing template. Additionally, the system includes fluorescentactivated cell sorting (FACS) means for selecting which microsphereshave a DNA sequencing template. Microfluidic distribution channel meansare used to distribute a selected microsphere with a DNA sequencingtemplate into a thermal cycling chamber. Autovalving means are used toensure that statistically only one microsphere will flow into onethermal cycling chamber. Sanger extension means, including the thermalcycling chambers, are used to produce thermal cycling extensionfragments from the microspheres carrying multiple copies of the DNAsequencing template. Integrated purification chamber means are used tocapture, purify and concentrate the extension fragments, and capillaryarray electrophoresis means are used to analyze the extension fragments.

In still another aspect, the invention is directed to a process forperforming sequencing. The process includes shearing DNA or RNA intofragments, ligating the fragments to form a mixture of desired circularand contaminating linear products, and selectively removing thecontaminated linear products. The process further includes generatingmicroreactor elements carrying multiple clonal copies of a sequencingtemplate, selecting which microreactor elements have a sequencingtemplate, and distributing the microreactor elements with the sequencingtemplate into thermal cycling chambers. Thermal cycling extensionfragments are produced from the microreactor elements carrying themultiple copies of the sequencing template. The extension fragments arecaptured, concentrated, and analyzed.

Various implementations of the invention may include one or more of thefollowing features. The microreactor element includes a microcarrierelement which carries the multiple copies of the clonal sequencingtemplate. The microreactor element is a bolus or a microemulsiondroplet. The microreactor element includes a microsphere carrying themultiple copies of the clonal sequencing template. The distributing stepis done such that only one microreactor element will pass into onethermal cycling chamber. An autovalve is used at an exit port of thethermal cycling chambers to ensure that only one microreactor elementwill flow into one thermal cycling chamber. The generating step includesgenerating multiple clonal copies of a sequencing template by emulsionPCR reactions or by a flow through PCR process. The selecting step isFACS.

In yet another aspect, the invention features a process for performingDNA sequencing. The process includes shearing DNA into DNA fragments,ligating the DNA fragments to form a mixture of desired circular andcontaminating linear products, and selectively removing thecontaminating linear products by exonuclease degredation. Microspherescarrying multiple clonal copies of a DNA sequencing template aregenerated by emulsion PCR reactions. The microspheres that have a DNAsequencing template are detected by FACS. The microspheres with the DNAsequencing template are distributed into thermal cycling chambers. Anautovalve at an exit port of a thermal cycling chamber is used to ensurethat statistically only one microsphere will flow into one thermalcycling chamber. Thermal cycling extension fragments are produced fromthe microspheres carrying multiple copies of the DNA sequencingtemplate. The extension fragments are captured, purified, concentratedand analyzed.

Various implementations of the invention may include one or more of thefollowing features. The capturing step uses an oligonucleotide capturematrix.

In still another aspect, the invention features a method for sequencing.The method includes receiving a microsphere carrying a clonal templateat an inlet port of a thermal cycling chamber and using a constrictionat an outlet port of the chamber to trap the microsphere in the chamberand substantially block further flow into the chamber.

In a further aspect, the invention features a method for analysisincluding producing a microsphere carrying a sequencing template. Themicrosphere is located in a thermal cycling chamber by use of aconstriction at an outlet port in the chamber such that further flowinto the chamber is substantially blocked.

Other implementations of the invention may include one or more of thefollowing features. A first valve is located at an inlet port of thethermal cycling chamber and a second valve is located at an outlet portof the thermal cycling chamber such that the second valve may be closedbefore the first valve. As such, a microsphere is moved out of theconstriction and into a main body portion of the thermal cycling chamberbefore thermal cycling.

The invention can include one or more of the following advantages. Thelaborious bacterial manipulations required by shotgun genome sequencingare replaced with easily automated and integrated in vitro steps. Assuch, millions of manual or robotic colony picking operations areeliminated. All fluidic and temperature control structures necessary toproduce, purify, and separate sequencing extension fragments areintegrated on a microfabricated device. Significant cost, time, andspace savings can be achieved. Other genetic analysis techniques can beperformed.

These and other features and advantages of the present invention will bepresented in more detail in the following specification of the inventionand the accompanying figures, which illustrate, by way of example, theprinciples of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The invention may best be understood by reference to the followingdescription taken in conjunction with the accompanying drawings thatillustrate specific embodiments of the present invention.

FIG. 1 is a diagrammatic representation of the steps involved in aconventional genome-scale sequencing operation.

FIG. 2 is a diagrammatic representation of the steps involved in agenome sequencing process in accordance with the present invention.

FIG. 3 is a diagrammatic representation of a cloning process that may beused with the present invention.

FIG. 4A is a diagrammatic representation of a single-channel microdevicein accordance with the present invention, FIG. 4B is an enlarged view ofa portion of the device of FIG. 4A, and FIG. 4C is a diagrammaticexploded view of the single-channel microdevice.

FIGS. 5A, 5B, and 5C are fluorescent images of a device in accordancewith the present invention during capture, wash, and sample injection,respectively, of extension fragments.

FIG. 6A is a diagrammatic representation of a sequencing reactionchamber of a device in accordance with the present invention, includingan enlarged view of a portion of the reaction chamber; FIG. 6B is a viewalong line 6B-6B of FIG. 6A; and FIG. 6C is a diagrammaticrepresentation of a constriction region of the reaction chamber of FIG.6A that is used to trap beads carrying the DNA to be sequenced.

FIG. 7A is a bright field image of a constriction region of an emptysequencing reaction chamber, FIG. 7B is a bright field image of aconstriction region of a sequencing reaction chamber filled withsolution but with no microsphere at the constriction region, and FIG. 7Cis a dark field image of a microsphere located at a constriction regionof a sequencing reaction chamber.

FIG. 8A is a diagrammatic representation of an alternate embodiment of aconstriction region of a sequencing reaction chamber designed for beadcapture, and FIG. 8B is a view along line 8B-8B of FIG. 8A.

FIG. 9A is a diagrammatic representation of one quadrant of anintegrated sequencing array system, and FIG. 9B is an enlarged view of aportion of the system of FIG. 9A.

FIG. 10A is a diagrammatic representation of a continuous flow throughPCR microfabricated device; FIG. 10B is an enlarged view at lines10B-10B of FIG. 10A, illustrating a “T”-injector region of themicrofabricated device; and FIG. 10C is an enlarged view at lines10C-10C of FIG. 10A, illustrating a portion of the channels of themicrofabricated device.

DETAILED DESCRIPTION

Reference will now be made in detail to some specific embodiments of thepresent invention including the best modes contemplated by the inventorfor carrying out the invention. Examples of these specific embodimentsare illustrated in the accompanying drawings. While the invention isdescribed in conjunction with these specific embodiments, it will beunderstood that it is not intended to limit the invention to thedescribed embodiments. On the contrary, it is intended to coveralternatives, modifications, and equivalents as may be included withinthe spirit and scope of the invention as defined by the appended claims.

In the following description, numerous specific details are set forth inorder to provide a thorough understanding of the present invention. Thepresent invention may be practiced without some or all of these specificdetails. In other instances, well known process operations have not beendescribed in detail in order not to unnecessarily obscure the presentinvention.

Furthermore, techniques and mechanisms of the present invention willsometimes be described in singular form for clarity. However, it shouldbe noted that some embodiments can include multiple iterations of atechnique or multiple applications of a mechanism unless notedotherwise.

The system and method of the present invention will be described inconnection with DNA sequencing. However, the system and method may alsobe used for RNA sequencing. Additionally, the system and method may beused for other genetic analysis of DNA or RNA.

The system and method of the present invention uses microreactorelements such as a microemulsion droplet or a bolus which, in someembodiments, include a microcarrier element such as a microsphere orbead. The microcarrier element, contained within a bolus or amicroemulsion droplet, is useful in capturing DNA or RNA products; thatis, amplified DNA or RNA can be chemically linked to the microcarrierelement. Alternatively, the amplified DNA or RNA may be carried by amicroreactor element without the use of a microcarrier element. Forinstance, a PCR reaction may be done in a bolus or a microemulsiondroplet, and then the resulting bolui or microemulsion droplets may berouted to the next step of the process.

A microfabricated integrated DNA analysis system (MINDS) of the presentinvention provides a system and method that are readily compatible withmicrofluidic sample component separation devices. As shown by FIG. 2,the MINDS process 200, in one embodiment, begins with the shearing ofDNA into DNA fragments (step 202). The fragments are then ligated toform a mixture of desired circular and contaminating linear products(step 204). The contaminating linear products are then selectivelyremoved, for instance, by exonuclease degradation (step 206). A colonyof microspheres carrying multiple clonal copies of a DNA sequencingtemplate is next formed (step 208). The colony may be generated, forexample, by emulsion PCR reactions. The microspheres having a DNAsequencing template are then identified (step 210). The microspheres maybe identified by a fluorescence activated cell sorting (FACS) technique.Only one such run is required, and it may take 6 hours or less tocomplete. The microspheres with the DNA sequencing templates are thendistributed into thermal cycling or sequencing reaction chambers whereextension fragments are produced; thereafter, the fragments are purifiedand concentrated (step 212). The extension fragment are then analyzed bya sample component separation apparatus, for example, a CE device (step214).

The MINDS method, process, and apparatus, in one embodiment, comprises amicrofabricated structure on which thermal cycling, affinity capture,sample purification, and capillary array electrophoresis (CAE)components are integrated. As will be discussed, such a system includesa microfluidic distribution channel to distribute microspheres carryingmultiple copies of a sequencing template into a plurality of thermalcycling or sequencing reaction chambers. An autovalve may be located atan exit port of the thermal cycling chambers to ensure that only onemicrosphere will flow into each chamber wherein thermal cyclingextension fragments are produced from the microsphere. Purificationchambers are connected to the thermal cycling chambers to capture,purify, and concentrate the extension fragments. Microfabricated CEseparation channels are connected to the purification chambers toanalyze the extension fragments.

The present invention eliminates the laborious, expensive, and timeconsuming in vivo cloning, selection, colony isolation, andamplification steps of conventional sequencing. Instead, these steps arereplaced with readily miniaturized and automated in vitro steps.

Microspheres are ideal carriers, providing flexible control over size,surface, fluorescent, and magnetic properties. Miniaturization of asequencing reaction chamber through microfabrication and the concomitantreduction in reagent volume makes possible the use of a single, clonalmicrosphere as a carrier for sufficient DNA sequencing template. Thisenables the use of a matched process flow that permits selection,amplification and sorting of clonal templates for direct integrationwith a nanoliter extension, clean-up and sequencing process.

FIG. 3 provides an overview of a cloning process 300 that may be usedwith the MINDS. The process includes library creation and selection. Inone embodiment, genomic DNA is isolated from whole blood and thensheared in a nebulizer generating DNA fragments (steps 302 and 304). TheDNA fragments are then subjected to an enzymatic treatment to yieldblunt-end DNA (step 306). Processed fragments are ligated into a vectoryielding a mixture of desired circular and contaminating linear products(steps 308 and 310). Exonuclease degredation then selectively removesthe insert and vector, the contaminating linear products, from theligation product so that greater than about 95% of the remaining vectorscontain inserts, the desired circular products (step 312).

The first five steps of the cloning process 300 (steps 302-310) followstandard library creation procedures. More specifically, Invitrogen(Carlsbad, Calif.) supplies a TOPO Subcloning Kit (#K7000-01) which iscapable of generating a 1 to 6 Kb insert genomic shotgun library in twohours. The subcloing kit requires about 20 micrograms (μg) of genomicDNA that can be obtained from 1 mL of whole blood using Qiagen's (Oslo,Norway) Blood & Cell Culture DNA Mini Kit (#13323). The DNA is shearedin a nebulizer generating 1-6 Kb DNA fragments. The fragments are thensubjected to an enzymatic treatment with T4 DNA and Klenow polymerasesand calf intestinal phosphatase (CIP) to yield dephosphorylatedblunt-end DNA. Processed fragments are next ligated into Invitrogen'spCR 4 Blunt-TOPO vector. Use of covalently linked vaccinia virustopoisomerase Ito the cloning vector results in rapid and efficientligation (a 5 minute room temperature ligation yields greater than 95%transformants) and permits dephosphorylation of the inserts prior toligation thereby preventing chimeric clone formation.

The final step in canonical library creation (step 312), which has beenmodified to be compatible with the in vitro MINDS process, is bacterialtransformation and antibiotic selection. Exonuclease degredationselectively removes insert and vector from the desired ligation product.Lambda exonuclease (New England BioLabs #M0262S) can be used for thisstep as it degrades 5′-phosphorylated and non-phosphorylateddouble-stranded DNA but is unable to initiate DNA digestion at the nickspresent in the vector after ligation. Assuming a pessimistic 1% recoveryof the starting genomic DNA, the resulting library contains greater than6,000 fold molecular excess needed for 10×sequence coverage, though thisexcess is reduced through dilution in the single-molecule PCRamplification step described later.

A gel separation and band purification step can be performed after DNAshearing (step 304) if strict control over insert size is required forpaired-end whole-genome de novo sequencing. Alternatively, insert sizecan be restricted using limiting extension times during PCRamplification or flow cytometric selection of microspheres within anarrow fluorescence intensity range. Labeling microsphere-clones with anintercalating dye, such as thiazole orange (TO), will yield afluorescent signal proportional to amplicon size. Since flow cytometryis a step in the MINDS process, this is an appealing size-selectionmethod that also eliminates the >5% recircularized vectors with the onlydrawback being reduced yield.

The vector used in the subcloning kit may be optimized for sequencingwith the T7 and T3 priming sites 33 by (base pairs) away from theinsertion site. Since the MINDS vector is free from biologicalconstraints, the sequencing priming sites can be moved closer to theinsert site and the pUC ori, fl ori, lacZ, and antibiotic resistancegenes can be removed. Optimized acrydite capture sequences and homo-PCRpriming sites can be inserted for improved sample clean-up and reducedprimer-dimer formation.

Sequencing in the MINDS process may use clonal template DNA attached toindividual microspheres or beads. The clonal attachment is accomplishedby linking one of the primers covalently to a microsphere.

Clonal isolation of 1,000 to 100,000 DNA molecules throughsingle-molecule amplification (for example microemulsion polonies) orcombinatorial hybridization approaches has been demonstrated. See,Mitra, R. D., et al., “Digital genotyping and haplotyping withplymearase colonies”, Proceedings of the National Academy of Sciences ofthe United States of America, 2003, 100(10): p. 5926-5931; Dressman, D.,et al., “Transforming single DNA molecules into fluorescent magneticparticles for detection and enumeration of genetic variations”,Proceedings of the National Academy of Sciences of the United States ofAmerica, 2003, 100(15): p. 8817-8822; and Brenner, S., et al., “Geneexpression and analysis by massively parallel signature sequencing(MPSS) on microbead arrays”, Nature Biotechnology, 2000, 18(6): p.630-634; each of which is hereby incorporated by reference. Conventionalmicrochip sequencing requires approximately 1 billion template DNAmolecules per reaction. Twenty billion extension fragments have beengenerated after 20× linear Sanger-sequencing amplification, representinga 20-fold excess for high signal-to-noise (S/N) detection using aconfocal radial scanner (1 million fragments per band X 1,000 bands=1billion molecules). This excess is necessary because a cross-injectionprocess requires streaming extension fragments across the separationcolumn until an equimolar mixture is obtained at the intersection andthen injecting about 5% of the total sample. An affinity capturedirect-injection strategy discussed below eliminates the 20-fold excessrequiring only 50 million initial template copies.

CPG Biotech (Lincoln Park, N.J.) produces a 5 micron (gm) controlledpore glass paramagnetic microsphere with a long chain alkylamine linkerfor covalent attachment to an amine modified primer that canconsistently bind 80 million DNA molecules. Starting from a single DNAmolecule, 30 cycles of PCR has a theoretical gain of 1 billion. Inpractice, the gain is lower due to less than 100% efficiency for eachcycle, reduced enzyme activity in later cycles, and limiting reagents inpL scale reactions. To generate clonal microspheres, a single DNAmolecule and a single primer-coupled microsphere must be placed in a PCRreactor. Efficient single-molecule PCR requires extremely small volumereactors (1-10 pL) to increase the effective concentration of a singleDNA molecule. Approximately 30 million microsphere clones are needed tosequence a human-size genome. High-throughput combination of a singlemicrosphere and DNA molecule in a chamber is possible usingstatistically dilute microemulsion solutions. If the DNA molecules andmicrospheres are each diluted such that one species is present in 10times the reactor volume, there is a high probability (greater than 99%)of concurrence in 1% of the reactors. Because 99% of the reactors arenon-productive, 100-fold more reactions (3 billion) are required togenerate 30 million microsphere clones.

Two possible approaches for generating large numbers of small volumereactions are microfabricated PCR devices and emulsion PCR. PCR devices,however, require thousands of runs to achieve the required 3 billionreactions. Emulsion PCR, on the other hand, has the ability to thermallycycle millions to billions of separate compartments in a single tubeusing a conventional thermal cycler. Clonal PCR amplicons attached tomagnetic particles using emulsion PCR have been produced. On average,each microsphere contained greater than 10,000 250 by amplicons, a valuebelow the theoretical maximum of 600,000 amplicons based on theavailable nucleoside triphosphates in each 5 μm diameter compartment.Fifteen micron microemulsion PCR compartments have been demonstrated inwhich the maximum number of 1,000 by amplicons is 5 million- a value tentimes less than the minimum required 50 million initial template copies.This problem may be solved in a variety of ways:

First, additional microemulsion PCR steps could be performed to increasethe amount of DNA linked to the beads. Second, an additionalnon-reagent-limited amplification step may be necessary after thefluorescence flow cytometry step. Secondary PCR amplification can beperformed in an on-chip, dual-use thermal cycling chamber in whichparamagnetic microspheres are routed to individual reactors andmagnetically retained. Fresh PCR mix is passed into the chambers andthermal cycled 15× to saturate each microsphere to the maximum of 80million templates. In this case, the magnetic field is maintained asSanger-sequencing reaction mix is washed into the chambers followed bysolid-phase sequencing. Finally, the S/N of the scanner may be improvedup to 10-fold enabling sequencing of about a 10-fold lower template thancalculated above.

Approximately 15 million 15 μm diameter compartments may be created in a25 μL aqueous phase, 75 μL oil phase emulsion. Three billioncompartments are required to generate 30 million coincident single DNAmolecule and microsphere events. Thus, approximately two 100 μL 96-wellplate reactions are required. When the reactions are complete, 30million microsphere clones must be separated from a background of 300million un-labeled microspheres.

As noted, the beads having sequencing templates may be identified byFACS. A system that may be used is the BD FACS ArrayBioanalyzer System,available from BD Biosciences, San Jose, Calif. The BD FACSArray flowcytometer can process up to 15,000 events per second, enabling theisolation of all clones needed for 10× coverage of a 3 billion basegenome in less than 6 hours. The beads will be treated with anintercalation dye, such as TO, that is nonfluorescent until intercalatedinto double-stranded DNA. The fluorescence intensity of TO is linearlyproportional to the amount of DNA allowing for easy differentiationbetween beads that have amplified DNA and those that do not.

A diagrammatic representation of a single-channel microdevice 400 inaccordance with the present invention is shown in FIGS. 4A, 4B, and 4C.The device may be fabricated as a four layer glass-glass-PDMS(polydimethysiloxane)-glass sandwich that incorporates microfluidicvalves, heaters, resistive temperature detectors (RTDs), and allreaction, capture and clean-up, and CE structures. The device, inanother configuration, may be fabricated as a glass-PDMS-glass-glassstack. A four layer microfabricated system including valves, heaters,RTDs, chambers, and CE structures is described in U.S. patentapplication Ser. No. 10/750,533, filed Dec. 29, 2003, entitled “FluidControl Structures In Microfluidic Devices”, assigned to the Assignee ofthe subject application, and which is hereby incorporated by reference.

The microdevice 400 is capable of performing all down-stream steps inthe MINDS process including extension fragment production, reactionclean-up, and extension fragment separation (steps 212 and 214 of FIG.2). The device 400 includes a thermal cycling or sequencing reactionchamber 402, a capture or purification chamber 404, and a CE system 406including separation channels 407. As shown, these components of thedevice 400 are connected by various valves and channels. A heater (notshown), for example, a kapton heater, may be used to heat the contentsof the thermal cycling chamber. The template of the chamber is monitoredby RTDs 405.

As shown in FIG. 4C, the device 400 includes three glass layers,including a channel layer 430, a via layer 432, and a manifold layer434. A PDMS membrane layer 436 is provided between the via layer 432 andthe manifold layer 434. The top layer 430 contains the thermal cyclingreactors, the capture chambers and the CE features. The second layer 432incorporates the RTDs on the top surface of the glass wafer and etchedfeatures on the bottom to form the valves and pumps with the membranelayer 436 below. The last layer 434 includes the heater, and completesthe valve and pump structures with pneumatic actuation lines anddisplacement chambers.

In one method of operation, a sequencing master mix is loaded from aport 408 of the device 400 through a microvalve 410 into the thermalcycling chamber 402. The volume of the chamber 402 may be approximately250 nano-liters (nL). The separation channels of the CE system 406 arefilled with linear polyacrylimide from a port 412 to a port 414. The CEsystem may be a 16-centimeter (cm) hyperturn system. An acrydite capturematrix is loaded from a port 416 to a port 418 to fill the capturechamber 404 and an intervening pinched chamber 419. After thermalcycling the chamber 402, extension fragments and residual reactants areelectrokinetically driven through a valve 420 and a channel 422 into thecapture chamber 404. The captured extension fragments are thenelectrophoretically washed and injected into a channel 424 forseparation by the CE system 406.

An advantage of this affinity capture, sample clean-up microdevice isthat purified extension fragments may be retained in 1-10 nL at aconcentration determined by the quantity of the acrydite monomer addedduring synthesis. FIGS. 5A, 5B, and 5C display fluorescence images(where relative applied potentials are indicated by “+” and “−”) fromthe operation of a microdevice 500 like that of the device 400 wherethermal cycling, capture, wash (purification), and concentration of asample as well as separation were performed. Specifically, an acryditecapture matrix was synthesized in a 2-mL solution of 5% w/v acrylamide,1× TTE (50 mM Tris, 50 mM TAPS free acid, 1 mM EDTA, pH=8.4), and 20nmol of the methacrylate-modified oligo(5′-Acrydite-ACTGGCCGTCGTTTTACAA-3′ (SEQ. ID NO. 1), TM=60.4 C, OperonTechnologies, Emeryville, Calif.). The solution was sparged with argonfor 2 hours prior to adding 0.015% w/v APS and TEMED to initiatepolymerization. The polymerized capture matrix was then loaded into acapture chamber 504 using a 1 mL syringe. A polymer sequencing matrix(CEQ, Beckman Corp., Fullerton, Calif.) was loaded into a CE system 506using a high-pressure gel loader. A C-track master mix was preparedcontaining 80 nM ET-primer, 1× C terminator mix (Amersham), and 4 nM PCRproduct and injected into a 250 nL thermal cycling chamber or reactor(not shown). Thermal cycling (35×, 94° C. 30 seconds, 45° C. 40 seconds,70° C. 40 seconds) was performed on-chip using a LabVIEW program(National Instruments, Austin, Tex.).

Sequencing reaction clean-up was performed by first equilibrating themicrodevice on a 50° C. heated stage for 30 seconds. Then, samplecapture (FIG. 5A) was initiated by applying 2000 V to the capturechamber outlet (port 416 of FIG. 4A) while grounding the reactor inlet(port 408 of FIG. 4A). Thus, the sample containing extension fragmentsand residual nucleotides, primers, and salts was electrophoreticallydriven from the thermal cycling chamber through a channel 522 into thecapture chamber 504. Extension fragments hybridize to the Acryditematrix in the capture chamber while residual reactants pass through.When oligonucleotide capture was complete, the retained extensionfragments were electrophoretically washed (FIG. 5B) for 30 seconds toremove excess primer and other contaminants still present in the capturechamber. After electrophoretic washing, the stage was ramped to 70° C.and equilibrated for 60 seconds to allow full denaturation of theproduct-matrix duplex. The denatured sample is directly injected intoseparation columns 507 of the CE apparatus 506 (FIG. 5C) by applying2,500 V to the anode while grounding the capture chamber outlet. (Imageshave been processed to highlight channel structure and removefluorescent surface contamination.)

Alternatively, a straight cross-injector and a 30-cm separationcapillary could be used. This will improve resolution.

The microsphere-colony creation procedure and a single reactormicrodevice can generate single-ended reads suitable for resequencingefforts. De novo whole-genome shotgun sequencing of complex genomesrequires paired-end reads from short and long insert clones. Long-rangePCR followed by fluorescent flow cytometry can be used on a subset ofmicrospheres to selectively generate long-insert clones. Traditionalpaired-end sequencing requires procedural and microdevice modificationssuch that clonal DNA could be released from the microspheres and routedto separate forward and reverse sequencing reactors. An alternativestrategy is to perform forward and reverse sequencing simultaneously ona subset of bases using an altered base labeling scheme. Thesepaired-end reads generate long single or double-base reads suitable foranchoring four-base single-ended reads in the sequence assembly.

As illustrated in FIGS. 6A, 6B and 6C, a microsphere 600 is used totransfer a clonal template into a thermal cycling or sequencing reactionchamber 602 where sequencing reactions are performed. This processrequires that one microsphere be introduced in each sequencing reactionchamber. Autovalving techniques for trapping an individual microspherein a sequencing reaction chamber, without the need for individuallyactuated valves and sensing, are used for this purpose.

As shown, the reaction chamber 602 includes an introduction channel orarm 604 and an exit channel or arm 606. The introduction channel is influid communication with an inlet port 605 of the reaction chamber,while the exit channel is in fluid communication with an outlet port 607of the reaction chamber. Self-valving is achieved by creating aconstriction or constriction region 608 at the outlet end or port 607 ofthe reaction chamber. The constriction region is semicircular in shape.

The constriction will trap a microsphere before it exits the chamber.The trapped microsphere will produce a drop in the flow rate, therebypreventing any further microspheres from flowing into the chamber. Thedilution of the microspheres will be chosen such that the probabilitythat another microsphere flows into the reaction chamber before thefirst microsphere has blocked the chamber is below about 0.5%. If thisstatistical approach is unreliable or leads to low sorting rates, anon-chip sensor for bead light scattering can be used, and two valves inthe sorting loop can be added to produce a more temporally uniform beaddistribution.

Glass fabrication processes can be used to create the device shown inFIGS. 6A-6C. In one example, all features except the constriction wereisotropically etched to a depth of 30 μm. The sample introduction andexit channels were set to 70 μm in width and 15 mm in length. Thesequencing reaction chamber had a volume of 250 nL. After the basicfabrication process was completed for the channels and the reactionchamber, a second mask was used to fabricate the constriction. For this,a more viscous photoresist (SJR 5740, Shipley, Marlborough, Mass.) wasspin-coated on a wafer at 2500 rpm for 35 seconds. This was followed bya soft bake at 70° C. for 7 minutes and 90° C. for 6 minutes. A higherviscosity photoresist gives more uniform coating on the featured wafer.The constriction pattern was transferred to the coated wafer using acontact printer. Alignment marks were used to align (+1 μm) theconstriction with the already etched channels and chamber. Afterdevelopment, the amorphous silicon masking layer was removed via plasmaetching, and the exposed glass was wet etched using 5:1BHF with aneffective etch rate of 2 μm/hour for 1.5 hours. This gave a constrictiondepth or height of 3 μm. The constriction width was set to 8 μm and thelength was set to 15 μm.

Testing of the self-valving concept was performed using 6 μm diameter,Streptavidin Coated Fluoresbrite YG Carboxylated Polystyrenemicrospheres (Polysciences Inc., Warrington, Pa.) suspended in asolution of 1× Tris (pH 8.0) and 1% Triton X-100 diluted to a finalconcentration of 1 microsphere/3 μL. Using the Poisson distribution, itwas calculated that a 10× dilution of the reaction chamber volume willinsure that the probability that another microsphere will enter the 250nL chamber before the first microsphere blocks the constriction is below0.5%.

A device like that shown in FIGS. 6A-6C was pre-filled with a solutionof 1× Tris (pH 8.0) and 1% Triton X-100. The microsphere solution (6 μL)was pipetted into an inlet access hole. A vacuum line was used to drawthe beads through the chamber. The pressure drop increased from -60 kPato -70 kPa when a microsphere was trapped at the constriction, andblockage occurred within the first 60 seconds. The experiment wascontinued for 12 minutes and no other microsphere was observed to enterthe chamber.

FIGS. 7A and 7B show bright field images of a constriction region 708 ofa thermal cycling chamber 702 at 20× magnification when empty and withsolution (but no microsphere), respectively. FIG. 7C shows a dark fieldimage of the thermal cycling chamber 702 with a fluorescent microsphere700 trapped at the constriction 708 and acting as a valve. Theexperiments were performed for microspheres of approximately 6 μmdiameter. The concept is easily scalable to larger microspheres toincrease the number of templates in the reactor. Also, smallermicrosphere could be used. The microspheres, in particular, may bebetween about 1 and 100 μm in diameter. In one embodiment, they areabout 10 μm in diameter.

An alternative autovalving embodiment, which will result in completeflow blockage, is presented in FIGS. 8A and 8B. Here, a reaction chamber802 has a near circular constriction or constriction region 808 formedat a chamber outlet port 807. The chamber and the constriction will bedouble-etched; that is, they will be etched on both of two joined glasssurfaces. This will produce a near circular constriction, as opposed toa semicircular constriction of the embodiment of FIGS. 6A-6C. This willprovide better valving for trapping a microsphere 800.

As discussed, valves will be incorporated on either side of a sequencingreaction or thermal cycling chamber 402 (see FIGS. 4A and 4B). Once amicrosphere has been trapped at the constriction, it needs to be pushedback into the chamber for thermal cycling so that there is goodaccessibility of the clonal templates to the polymerase, primers, dNTPsand ddNTPs. By closing the valve 420 at the outlet of the chamber beforeclosing the inlet valve 410, the microsphere will be pushed back intothe chamber as a result of the finite dead volume of the valves. Thisprinciple is easily scaled to an array system by actuating the inlet andoutlet valves in parallel.

To realize a high-throughput and fully integrated system for DNAanalysis, a microfabricated array of integrated analyzers thatincorporate thermal cycling chambers, purification chambers as well asseparation channels for CE analysis of the sequencing fragments isprovided. A schematic of such a system 900 is shown in FIGS. 9A and 9B.This system extends the single channel device of FIGS. 4A-4C to an arraystructure that is able to perform highly parallel analyses.

According to various embodiments, the system 900 includes multiplethermal cycling chambers 902 and associated sample purification orcapture chambers 904 arranged about a circular axis to form a radiallyparallel system. The thermal cycling chambers and the samplepurification chambers are all integrated with a CE analyzer system 906including separation channels or microchannels 908. The CE analyzer hasa common central anode (A) 910, a cathode reservoir (C) 912, and a wastereservoir (W) 914. The cathode and anode reservoirs are associated withadjacent sets of separation channels 908. The microchannels 908 areconnected to the anode 910 for detection using a rotary confocalfluorescence scanner of the type discussed in the article entitled:“High-throughput genetic analysis using microfabricated 96-samplecapillary array electrophoresis microplates”, Peter C. Simpson, et al.,Proc. Natl. Acad. Sci. USA, Vol. 95, pp. 2256-2261, March 1998, which ishereby incorporated by reference.

The system 900 further includes a distribution channel 916, integratedheaters 918 a and 918 b, and RTDs 920. The heaters 918 a and 918 baddress the thermal cycling chambers and purification chambers,respectively, in parallel. As such, the use of simple ring heaters todrive the thermal cycling and sample purification reactions is more thanadequate.

The temperature of these reactions are monitored by the RTDs. In oneembodiment, four-equally spaced RTDs are integrated on the substrate toprovide precise temperature sensing across the heater 918 a for optimalthermal cycling performance. Similarly, four-equally spaced RTDs may beused to monitor the temperature of the heater 918 b.

The system 900 also includes an array of integrated valves and ports forcontrolling the system flow process. The valves may be monolithicelastomer (PDMS) membrane valves. The system may be fabricated asdescribed in the above-identified U.S. patent application Ser. No.10/750,533, which has been incorporated by reference. As such, thesystem may include a four layer glass-glass-PDMS-glass stack thatincorporates the microfluidic valves and pumps, the RTDs, the thermalcycling chambers, the clean-up and concentration chambers, and the CEchannels.

The system 900, in one embodiment, includes 24 thermal cycling chambers,24 purification chambers, and 24 CE channels arranged on a quadrant of a150-mm diameter glass substrate. Each thermal cycling chamber (˜250 nL)is isolated from the distribution channel by PDMS membrane valves (V1)922. Rigid containment of the chamber volume with active valves isnecessary for bubble-free loading and immobilization of a sample duringthermal cycling since sample movement, bubble formation, and sampleevaporation can seriously affect the performance.

The RTDs may be fabricated of titanium (Ti) and platinum (Pt). Differentmaterials (Au, Al, Pt, Ni) using different metal deposition techniques,such as sputtering and both thermal and electron-beam evaporations, maybe used to fabricate the heaters. Nickel heaters exhibit good heatinguniformity, have good scratch resistance, show no noticeable degradationof performance even after hundreds of thermal cycles, and are easilyfabricated. The microfabricated heaters and the thermal cycling chambersare positioned away from the CE microchannels to avoid evaporation ofbuffer in the cathode reservoirs and to minimize the heating of theinjection region during thermal cycling. The resistive ring heaters canbe fabricated on the back side of the bottom wafer to ensure goodthermal transfer between the heaters and the chambers. Theequally-spaced RTDs are integrated on the microplate to provide precisetemperature sensing ensuring temperature uniformity across the heatersfor optimal performance.

The thermal cycling chambers may be cycled with a LabVIEW program(National Instruments, Austin, Tex.). (LabView VI) Temperature controlcan be accomplished through a proportion/integration/differentiator(PID) module within the LabVIEW program.

In operation, in one embodiment, a sequencing separation matrix isintroduced into the CE separation channels using positive pressurethrough the anode 910. A separation matrix fills the cathode reservoirs912 and the waste reservoirs 914, as well as arms 924 connecting thecross-injection point to the associated sample purification chambers.The filling rate can be modulated through adjustment of the widths andlengths of the various interconnecting channels. The cathode and anodereservoirs, and the connecting arms may be filled at the same rate.Water, for example, is loaded from a loading (L) port 926 connected tothe distribution channel 916 to fill the thermal cycling chambers 902and the sample purification chambers 904 ensuring continuity forsubsequent processes. An exit port (E) 936 is shared by both the thermalcycling chambers 902.

A capture gel matrix is loaded into the sample purification chambersthrough ports (F) 928. Libraries of clonal DNA beads that have beensorted and prepared using a FACS unit are loaded from the port 926(valves (V1) 922 and (V2) 930 open, and valve (V3) 932 closed) using,for example, a syringe pump. A pressure transducer is employed in theinlet to monitor the head pressure. Auto-valves at the exit ports of thethermal cycling chambers will stop the flow into a chamber once a beadis trapped in the auto-valve as described above. As each auto-valve isfilled with a bead in each thermal cycling chamber, the head pressurewill continue to increase. In this manner, loading a bead in eachthermal cycling chamber is achieved by monitoring the head pressure.

Upon filling the chambers, the valves (V2) 930 and (V1) 922 are closedand thermal cycling reactions initiated. Since the beads will be sorteddirectly in the thermal cycling cocktail, no additional steps are neededbefore thermal cycling. Alternatively, sorting could be performed in abuffer followed by introduction of the thermal cycling cocktail into thethermally cycling chambers to minimize reagent usage. The heaters 918 aand 918 b, as noted, drive the thermal cycling and sample purificationreactions, and the reaction temperatures are monitored by the RTDs 920.

Thermal cycling products are driven to the purification chambers byapplying an electric field across the port (L) 926 and a sample port orelectrical contact point (S) 934 with the valves (V1) 922 and (V3) 932open. Flushing of the non-captured reagents is performed by applying anelectric field between the port (S) 934 and the port (F) 928.Thereafter, the sequencing products are injected from the purificationchambers into the associated microchannels 908 for CE separation, byapplying a potential between the port (S) 934 and the waste reservoir(W) 914. The rotary confocal fluorescence scanner is used to detect thesequencing samples. The scanner interrogates the channels sequentiallyin each rotation of the scanner head.

As shown, separations of the purified DNA sequencing products areachieved by using cross-injection. Alternatively, direct injection couldbe used.

In another embodiment, the microemulsion PCR approach of colonyformation is replaced by a continuous flow through PCR technique inwhich bouli or droplets for performing single-molecule PCR amplificationof templates are formed and then amplified. The bouli or plugs areformed, for example, by combining an aqueous solution of PCR reagentsand dilute template fragments with a hydrophobic solution that acts as acarrier. The bolui produced by this technique will contain statisticallya small number of copies of a sequencing template. Ideally theconcentration is chosen so that only one in 10 bolui contain a singlesequencing template. This mean that only one in 100 will simultaneouslycontain undesirably two templates. A microsphere or bead carrying one ofthe primers in the PCR reaction can also be entrained within a bolus ordroplet in the process of performing this technique. Thus, a colony maycomprise bolui carrying multiple clonal copies of a single sequencingtemplate or bouli having microspheres carrying such copies.

As shown in FIGS. 10A, 10B, and 10C, a continuous flow throughmicrofabricated PCR system 1000 includes ports 1002 and 1004. An aqueoussolution of microspheres, PCR reagents and template fragments, in oneembodiment, is introduced into a “T”-injector region 1006 via the port1002, while a hydrophobic carrier solution is introduced into the“T”-injector region via the port 1004. A bolus or droplet 1012containing a microsphere is thus formed downstream of the “T”-injectorregion.

The system 1000 further includes an optical sensor 1007 (FIG. 10B)located downstream of the “T”-injector near an input end of a channel1014. The sensor may comprise a focused laser beam and a photodiode todetect off-axis light scattering.

The sensor is configured to select a bolus that contains a bead and toreject bolui with no bead or with more than one bead. A rejected bolusis passed out of the system via a valve 1009 and a waste port andchannel 1011. The valve 1009 may be a four-layer PDMS valve that whenopen allows a rejected bolus to exit the system via the waste port 1011.The valve may be activated via a valve actuation port 1013. Since thefluidic resistance of the continuous flow system is so great, no valveis needed in the main channel 1014.

The system 1000 is designed for two-step PCR where the anneal and extendsteps are combined. As such, the system includes two temperature zones1008 and 1010 through which droplets 1012 pass via the flow throughchannels 1014. The device 1000 includes RTDs 1016 and associated RTDcontact pads 1018 for monitoring the temperature of the differenttemperature zones. A bolus and microsphere, after undgergoingtwo-temperature PCR, exit the system via an exit port 1020.

As discussed, the microsphere can be treated with an intercalation dye,such as TO, that is nonfluorescent until intercalated intodouble-stranded DNA. Therefore, the microspheres that have amplified DNAcan be identified by, for example, the FACS technique after they passthrough the port 1020. Thereafter, the microspheres are introduced intoa distribution channel of a device like the device 900 (See FIGS. 9A and9B). The microspheres that do not have amplified DNA, on the other hand,will be disposed of.

The continuous flow through PCR system 1000, in one example, usesnanoliter droplets of PCR reagents suspended in a perfluorohydrocarboncarrier. See, Song, H., et al., “A microfluidic system for controllingreaction networks in time”, Angewandte Chemie-International Edition 42,768-772 (2003), which is hereby incorporated by reference. Dropletscontaining microspheres, PCR reagents, and template fragments, separatedfrom one another by an immiscible liquid, are formed at themicrofabricated T-injector 1006. Thereafter, the droplets flow throughthe regions 1008 and 1010 of the device that are held atanneal-extend-denature temperatures. The lengths of the channels 1014 inthe different temperature zones 1008 and 1010 are selected based onrequired residence times at those temperatures. The cross-section of thechannels and the desired flow rates are chosen to achieve stable dropletformation. The parameters of the device may be set to achieve a hotstart at the denature temperature for 90 seconds, followed byanneal/extend for 45 seconds with a 1 second auto-extend for 35 cyclesand a denature time of 15 seconds. The last 15 cycles may be configuredto have a constant anneal/extend time of 80 seconds.

The device 1000 may comprise two 1.1-mm thick by 10-cm diameterborofloat glass wafers. The channels on the patterned wafer may have aD-shape cross-section with a width of about 210 μm and a depth of about95 μm. On the other wafer, Ti/Pt RTDs may be fabricated for monitoringtemperature gradients at different points in the device. Holes may bedrilled for accessing the channels, and the two wafers may be thermallybonded to each other to form enclosed channels. The nanoports 1002 and1004 are used to interface the device with micro-liter syringes throughPEEK tubing. The glass surface of the channels may be renderedhydrophobic through silanization with1H,1H,2H,2H-perfluorodecyltrichlorosilane, based on a procedurepublished by Srinivasan et al., “Alkyltrichlorosilane-basedself-assembled monolayer films for stiction reduction in siliconmicromachines”, Journal of Microelectromechanical Systems 7, 252-260(1998), which is hereby incorporated by reference. Two syringe pumps maybe used to dispense water and a 10:1 mixture of perfluorodecalin(mixture of cis and trans, 95%, Acros Organics, N.J., USA) and1H,1H,2H,2H-perfluorooctanol (Acros Organics, N.J., USA) at a flow rateof 0.5 μl/min each. FIG. 10B shows the droplet formation process at theT-injector at about 1 droplet/s, and FIG. 10C shows the droplets as theymove through the channel.

The technique, in one embodiment, uses microbeads in 10 nL droplets.Throughput is maintained at one in ten droplets containing both a singletemplate molecule and a single bead. The aqueous PCR mix is preparedsuch that there is one template molecule per 100 nL of mix,corresponding to one molecule for every ten droplets. Also in the PCRmix are microbeads at a concentration of one per 10 nL of mix,corresponding to one bead for every droplet on average. As dictated bythe Poisson distribution, 37% of the 10 nL droplets will contain onlyone microbead, the remaining containing either none or two plus. Eachdroplet, as discussed, is then optically scanned to determine the numberof beads it contains. If the droplet does not contain a singlemicrobead, the valve 1009 is opened, passing the droplet to waste.Approximately one third of the droplets will contain a single bead andare routed to the main channel; thus, the average flow rate is equal toabout 1 droplet every 1.5 s. As such, every droplet in the main channelcontains a single microbead, and one in ten also contains a singletemplate molecule.

At the end of the device 1000, the droplets may be collected at the port1020 via a standard capillary into a microfuge tube. The droplets arebroken through centrifugation. Microbeads are collected and washed in 1×TE, again through centrifugation. The microbeads are routed into thethermal cycling chambers of the device 900 by either autovalving oractive valving with on-chip detection. On-chip detection may comprisethe use of an optical scanner or a timing arrangement that determineswhen a microsphere is located adjacent to an inlet of a thermal cyclingchamber. The optical scanner, as discussed, may use bead lightscattering to determine the location of a microsphere within thedistribution channel. The timing arrangement is based on the fact thatthe fluid in the distribution channel is incompressible. As such, thelocation of a microsphere within the distribution channel, for instance,adjacent to an inlet of a thermal cycling chamber, can be calculatedfrom the time that the microsphere has been in the distribution channel.This is advantageous because the valved entrances to each of the 96inputs from the distribution channel, for example, can be actuated by asingle pneumatic input. Also the pneumatic input is not actuated until adetected bead is flowed directly opposite a reactor that does not have abead yet.

To demonstrate the feasibility of generating sequencing templates usingtwo-temperature PCR, high-temperature primers were designed and testedin a conventional thermal cycler. Two primers,M13_(—)2T_F-5′TTCTGGTGCCGGAAACCAGGCAAAGCGCCA-3′(SEQ. ID NO. 2) Tm=70.3°C. and M13_(—)2T_R 5′-ACGCGCAGCGTGACCGCTACACTTGCCA-3′ (SEQ. ID NO. 3)Tm=70.7° C. were designed to generate a 943 by amplicon from the M13genome. To approximate the concentration of a single template moleculein a 11 nL emulsion compartment, 18.75 femtograms of M13 template werecycled in a 25 uL PCR reaction (94° C. 1.5 min followed by 50 cycles of94° C. 10 s, 70° C. 30 s with an auto-extend of 1 s/cycle). Theresulting amplicon was a single clean peak at the expected size with ayield of about 40 ng/uL.

If such a system is used to amplify genomic DNA, for 1× coverage andaverage fragment size of 1000 bps, about 3 million fragments need to beamplified. The probability that two different fragments end up in asingle droplet can be reduced to less than 0.01 by diluting thefragments in the PCR reagent such that on an average one in ten dropletscontains a fragment. Hence, the system will have to process 30 milliondroplets. This device is designed to generate one droplet about every1.5 seconds. If 20 such devices are run in parallel, the entire genomecan be amplified and interfaced with a bank of the sequencing systems900 to produce 1× coverage in only one month.

While the invention has been particularly shown and described withreference to specific embodiments, it will also be understood by thoseskilled in the art that changes in the form and details of the disclosedembodiments may be made without departing from the spirit or scope ofthe invention. For example, the embodiments described above may beimplemented using a variety of materials. Therefore, the scope of theinvention should be determined with reference to the appended claims.

1. A process for performing sequencing comprising: shearing DNA or RNAinto fragments; ligating the fragments to form a mixture of desiredcircular and contaminating linear products; selectively removing thecontaminating linear products; generating microreactor elements carryingmultiple clonal copies of a sequencing template; selecting whichmicroreactor elements have a sequencing template; distributing themicroreactor elements with the sequencing templates into thermal cyclingchambers; producing thermal cycling extension fragments from themicroreactor elements carrying multiple copies of the sequencingtemplate; capturing and concentrating the extension fragments; andanalyzing the thermal cycling extension fragments.
 2. The process ofclaim 1 wherein the microreactor element includes a microcarrier elementwhich carries the multiple copies of the clonal sequencing template. 3.The process of claim 1 wherein the microreactor element is a bolus or amicroemulsion droplet.
 4. The process of claim 3 wherein themicroreactor element includes a microsphere carrying the multiple copiesof the clonal sequencing template.
 5. The process of claim 1 wherein thedistributing step is done such that only one microreactor element willpass into one thermal cycling chamber.
 6. The process of claim 5 furtherincluding using an autovalve at an exit port of the thermal cyclingchambers to ensure that only one microreactor element will flow into onethermal cycling chamber.
 7. The process of claim 1 wherein thegenerating step comprises generating multiple clonal copies of asequencing template by emulsion PCR reactions or by a flow through PCRprocess.
 8. The process of claim 1 wherein the selecting step isfluorescence activated cell sorting (FACS).
 9. A process for performingDNA sequencing comprising: shearing DNA into DNA fragments; ligating theDNA fragments to form a mixture of desired circular and contaminatinglinear products; selectively removing the contaminating linear productsby exonuclease degradation; generating microspheres carrying multipleclonal copies of a DNA sequencing template by emulsion PCR reactions;selecting which microspheres have a DNA sequencing template byfluorescence activated cell sorting (FACS); distributing themicrospheres with the DNA sequencing template into thermal cyclingchambers; using an autovalve at an exit port of a thermal cyclingchamber to ensure that statistically only one microsphere will flow intoone thermal cycling chamber; producing thermal cycling extensionfragments from the microspheres carrying multiple copies of the DNAsequencing template; capturing, purifying and concentrating theextension fragments; and analyzing the thermal cycling extensionfragments.
 10. The process of claim 9 wherein the capturing step uses anoligonucleotide capture matrix.
 11. A process for use in sequencingcomprising: receiving a microsphere carrying a clonal template at aninlet port of a thermal cycling chamber; and using a constriction at anoutlet port of the chamber to trap the microsphere in the chamber and tosubstantially block further flow into the chamber.
 12. A method foranalysis comprising: producing a microsphere carrying a sequencingtemplate; and locating the microsphere in a thermal cycling chamber byuse of a constriction at an outlet port in the chamber such that furtherflow into the chamber is substantially blocked.
 13. The method of claim12 wherein a first valve is located at an inlet port of the thermalcycling chamber and a second valve is located at an outlet port of thethermal cycling chamber such that the second valve may be closed beforethe first valve to move a microsphere out of the constriction and into amain body portion of the thermal cycling chamber before thermal cycling.